ABSTRACT: TGF-? superfamily members play a central role in remodeling of the infarcted heart, modulating phenotype and function of all cell types involved in myocardial inflammation, repair, and fibrosis. TGF- ?s function by activating molecular cascades involving Receptor-activated Smads (R-Smads), or through Smad-independent mechanisms. However, effective repair requires tight regulation of TGF-? actions, in order to prevent adverse consequences that lead to maladaptive cardiac remodeling. Overactive, or prolonged TGF-? signaling can promote cardiomyocyte, fibroblast and macrophage activation, stimulating persistent fibrotic, hypertrophic and phagocytic responses. The endogenous mechanisms responsible for negative regulation of TGF-? responses in tissue repair, remodeling and fibrosis remain unknown. The current proposal explores the molecular mechanisms responsible for suppression and termination of TGF-? superfamily signaling in the infarcted and remodeling myocardium. Our unpublished preliminary data suggest an important role for 2 distinct mechanisms in negative regulation of TGF-? superfamily signaling. First, cell-specific induction of the inhibitory Smads (I-Smads), Smad7 and Smad6 may negatively regulate TGF-? and BMP responses following myocardial infarction. Second, TGF-? signaling may be regulated at the receptor level through cell-specific induction of the transmembrane pseudoreceptor BAMBI (BMP and activin membrane-bound inhibitor), which lacks an intracellular kinase domain, and inhibits TGF-? signaling, at least in part, through interactions with I-Smads. The role of the cell-specific mechanisms for negative regulation of TGF-? will be explored in 4 specific aims: Specific aim 1: to investigate the role of Smad7 in regulation of cardiomyocyte, fibroblast and macrophage phenotype following infarction. Our preliminary studies show that Smad7 is markedly upregulated in border zone cardiomyocytes, and in infarct myofibroblasts and macrophages, and that Smad7 knockdown or overexpression modulate TGF-?-driven cellular responses. Accordingly, we will use cardiomyocyte-, fibroblast/myofibroblast- and myeloid cell-specific Smad7 knockout mice, recently generated by our laboratory to explore the cellular effects of Smad7 on the infarcted heart. Specific aim 2: to dissect the molecular mechanisms responsible for the effects of Smad7 in vivo and in vitro. Smad7 actions may involve suppression of Smad-dependent or non- Smad pathways and may involve interactions with T?Rs, R-Smads or TGF-?-independent signals. Our preliminary studies in cardiac fibroblasts suggest that Smad7 restrains Smad2/3 activation without affecting phosphorylation of T?Rs. The molecular mechanisms for Smad7-dependent regulation of cardiomyocyte, fibroblast and macrophage phenotype will be studied in vitro and in vivo, using both loss and gain-of-function approaches. Specific aim 3: to investigate the role of Smad6 in repair and remodeling of the infarcted heart. Our preliminary studies show Smad6 induction in cardiomyocytes, fibroblasts, and macrophages infiltrating the healing infarct, and demonstrate that fibroblast Smad6 exerts actions distinct from Smad7-mediated effects. Conditional Smad6 knockout mice will be generated to dissect the cell- specific actions of Smad6 in the infarcted and remodeling myocardium, and the mechanisms responsible for Smad6-mediated effects will be explored in vivo and in vitro. Specific aim 4: to study the role of BAMBI in regulation of TGF-? responses in the infarcted and remodeling heart. Our preliminary studies show that BAMBI is upregulated in infarct myofibroblasts and macrophages. Accordingly, we will use fibroblast- and macrophage-specific BAMBI knockouts and in vitro experiments, in order to study the role of BAMBI in modulating cell-specific TGF- ? actions in the infarcted heart. Moreover, we will explore interactions between BAMBI and the I- Smads. The proposed studies will provide the first systematic investigation of the mechanisms responsible for negative regulation of TGF-? in a model of tissue injury.